(1) Field of the Invention
The present invention relates to diagnosis using DNA and an assay for properties of DNA as well as a method for DNA analysis or assay.
(2) Description of the Related Art
It has been becoming popular to use DNA for diagnosis of disease. In such diagnosis of disease, (1) a DNA probe having a complementary sequence to a target DNA is prepared and a probe assay is carried out to see if this DNA probe hybridizes with the target DNA; (2) a certain region of sequence coding for a target DNA is chosen and subjected to polymerase chain reaction amplification using two DNA probes (primers), the resulting DNA fragment is read or its length is examined, and based on the DNA fragment information obtained, assay is performed; etc. The thus obtained results are utilized for diagnosis or the like. These methods are applicable to analysis or assay for a single kind of DNA, at most, a few kinds of DNAs but not appropriate for DNA assay of a vast number of DNA fragments or total evaluation of long DNA.
However, DNAs or genomes in vivo function while interacting with each other. It is, therefore, strongly desired to collectively assess chromosomes or all DNAs contained therein. For example, in a spotlighted cDNAs among the genome-project, it has been attempted to detect the kind and amount of cDNA complementary to mRNA thereby to collectively grasp an organism, paying attention to the mechanism of DNA functioning in the organism that, where DNA functions in an organism, DNA information is first transcribed onto mRNA and a protein is synthesized based on the information to function the organism. In this attempt, cDNAs are fished out of a vital sample and the respective cDNAs are sequenced to analyze the frequency of each CDNA appearing in one tissue (body mapping).
The body mapping involves the following procedures. First, cDNA is prepared from mRNA (in a mixture of diverse cDNAs) and then cloned. E. coli containing cDNAs is spread and cultured on an agarose plate to obtain colonies, each of which contains one of the desired cDNAs. The desired cDNA is taken out and sequenced to identify the kind of cDNA. In a similar manner, a desired cDNA is taken out of each colony and sequenced, whereby the same cDNA often appears. When attention is given to one particular cDNA present in one tissue, the larger the amount of this particular cDNA, the more likely the particular cDNA corresponds to the gene strongly expressed in the tissue and as the result, the higher the frequency of the gene appearance in the colony. Accordingly, there is proposed a method for determining the frequency of CDNA appearance which comprises performing cDNA sequencing in many colonies to see how many times a particular cDNA appears in the colonies (Katsuji, Murakawa et al., Genomics, 23, 379-389 (1994)).
On the other hand, another attempt for DNA diagnosis has also been proposed, paying attention to a genome (DNAs in all chromosomes) or the entire profile of a particular chromosome. A fingerprinting technique called gene scanning, which is also called Landmark genome scanning (LGS method), involves the steps of selectively digesting DNA with 8 base cutter restriction enzyme (which digests once per 48 to 64 kbs) such as Not I, etc., binding the digested fragments to a radioisotope tag or a nucleotide labeled with fluorophore, separating the fragments by electrophoresis, then cutting the DNA fragments in a gel with a 4 base cutter restriction enzyme (which digests once per 44 to 256 bases), and subjecting the DNA fragment on the upper end of a polyacrylamide slab gel to two-dimensional electrophoresis. The thus obtained pattern is utilized as a fingerprint, thereby to comprehend the entire profile of DNA. An attempt includes use of the pattern for diagnosis, noting that a DNA pattern in normal cells is different from that of abnormal cells suffered from cancer, etc.
However, a good technique is not found so far, since it should be examined in the foregoing methods in which particular site of long DNA there is abnormality.
It is important for early detection of diseases or understanding of DNA function in cells to examine a long and large DNA or clarify the entire profile of a sample containing diverse cDNAs. As stated above, however, any good technique sufficient for the purpose has not been developed yet. According to the techniques explained hereinabove, it is necessary to determine the base sequences of very many clones. Much labor and time required make it impossible to practically apply these techniques to various samples. Conventional DNA probing is only enough to examine, at best, several to several ten kinds of DNAs in one cycle of operation but not suitable for assaying cDNAs or DNA fragments of several hundreds to thousands in one cycle of operation. In addition, the cDNA analysis methods described above are not applicable to detection of long DNA where abnormality is located.
On the other hand, the gene scanning technique can meet the foregoing requirements but encounter problems that a huge amount of enzymes are consumed in the second digestion with a restriction enzyme and in the two dimensional electropherogram, the abscissa which is a scale for length of the DNA fragment occurred in the first digestion and the ordinate which gives some scale for length of the ultimate fragment are not always quantitative so that it is difficult to construct a database with these data.